Short interfering RNAs (siRNA) as gene-specific therapeutic molecules are resourceful tools to accurately control gene expression. However, delivery of these molecules to specific tissues is confronted due to their anionic nature, large size and non-specific effects preventing their clinical utility. Additionally, the delivery of siRNA to the brain parenchyma is restricted by the blood-brain barrier hampering treatment of subjects experiencing neurodegenerative conditions having long-term effects. To overcome these limitations while exploring for selectivity and non-toxicity, many peptide carriers were earlier established by conjugating antibody ligands and fusion proteins to nanoparticles through genetic engineering approaches, with the objective of targeting transferrin and insulin receptors of endothelial cells lining the brain capillaries. In a prominent advancement, different from these studies, numerous cell-targeting peptides were chosen through phage display by virtue of inherent tropism, increased avidity to mammalian cell surface receptors and ease of production. In an equivalent strategy, peptides with the binding characteristics of tetanus toxin to trisialoganglioside GT1b led to the discovery of a 12-aa peptide Tet1, offering the possibility of conjugating short peptides to larger protein scaffolds to generate multi-functional fusion proteins with neuronal tropism. Succeeding these studies, in a similar effort, Georgieva et al. developed strategies to conjugate neurotropic peptides to lipid-based molecules to impart in vivo stability, which demonstrated remarkable transcytotic capacity in vitro suggesting an identical mechanism in vivo. Upon systemic delivery, the targeting molecules, having affinity for GM1, were found to localize in the brain parenchyma and additionally in the lungs mandating further explorations to understand the potential of derivatized polymers displaying broad selectivity. Majority of non-viral vectors for nucleic acid delivery were earlier developed using the cationic lipids, cationic cell-penetrating peptides, and dendrimers. Spontaneous interaction of these molecules with nucleic acids led to the formation of stable non-covalent complexes. Knowledge-based rational design strategies subsequently led to the usage of multiple components for superior delivery and stability leading to successful target-specific gene silencing. Taking cues from neurotropic viruses, Kumar et al., fused the arginine peptide (9-mer) to a peptide derived from rabies virus glycoprotein (RVG) to facilitate electrostatic interaction with siRNA and specifically target acetylcholine receptors. The synthetic peptide fusion RVG-9R facilitated transvascular delivery of siRNA resulting in target specific gene silencing. However, presence of high density cationic charge on the carrier could lead to the formation of heterogeneous particles, non-specific biodistribution and lower yield of protein in suitable host systems.
RNA-recognition motifs conserved among double-stranded RNA-binding proteins lend their attributes to the design of modular fusion proteins. In a study using an arginine-rich peptide, tandem repeats of TAT was fused to the double-stranded RNA Binding Domain (DRBD) to electrostatically bind siRNA. The approach, although facilitating the delivery of siRNA into several primary cells including glioma lacked cell-specificity. It is thus evident that multiple DRBD motifs fused with cationic peptides may not confer additional in vivo advantage due to their likely interaction with serum proteins, lack of target selectivity and tendency to aggregate. Versatility of TARBP2 fusion protein whose conformation-dependent binding to double-stranded RNA abolished the prerequisite of positively charged peptides. The strong binding interactions led to the formation of neutral nanosized complex which were stable upon systemic administration. In the present invention, we have overcome the aforesaid barriers and invented a method to deliver siRNA selectively to target the brain and central nervous system, by fusing the RNA-binding domain with a peptide having sequence GGGGHLNILSTLWKYRC represented by SEQ ID NO. 9 that can retain flexibility and at the same time target GM1 and GT1b expressing cells, by virtue of their natural abundance of these receptors in neuronal cells to permit accumulation of the silencer complex with the aim of targeting disease causing genes in neurodegenerative condition by the advantage and ability of the peptide chimera to cross the blood-brain barrier by receptor-mediated transcytosis by mediating efficient and effective RNAi via GM1 in brain tissue.